Production method of recombinant rotavirus structural proteins and vaccine composition

ABSTRACT

The present invention relates to a method for producing recombinant human rotavirus structural proteins by culturing transformed plant cell, and to a vaccine composition comprising the same as an effective component. More specifically, the method comprises the steps of preparing the recombinant expression plasmid comprising cDNA fragment encoding human rotavirus structural protein; transforming plant cell with the expression plasmid; and recovering the vaccine composition including the rotavirus antigen from cell culture. The method for producing the recombinant human rotavirus structural proteins has advantages in higher yield of more than 0.3 mg/L, lower cost and lower contamination hazard than those of the eukaryotic expression system such as baculovirus and animal cell, and can be used for producing edible vaccine. Also, the invention shows that the human rotavirus structural protein can be produced with the high aspect rotary vessel reactor.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for producing thestructural proteins of the recombinant rotavirus by culturing atransformed plant cell, and the vaccine composition comprising suchproteins as an effective component. More particularly, the methodcomprises the steps of producing the recombinant expression plasmidincluding the gene encoding the human rotavirus structural protein,transforming the plant cell with the expression plasmid, cultivating theplant cell, and obtaining the vaccine composition including rotavirusantigen recovered from the culture solution.

BACKGROUND OF THE INVENTION

[0002] In 1973, Bishop discovered the Rotavirus in Australia firstly.The rotavirus is a double stranded RNA virus and belongs to theReoviridae family. The rotavirus causes acute gastroenteritis in infantand is infected via fecal-oral route after an incubation period of about1 to 3 weeks. The disease is severe in 6 to 24 week infant, but is mildor asymptomatic in neonate or most of adults. Thus, the acute infectiousdiarrhea by the rotavirus is a main cause of the death in the world.Moreover, it is estimated that about a million of patients are died ofthe infectious diarrhea by the rotavirus in the developing countries[see reference: Blacklow, N. R. & Greenburg, H. B., (1991) Viralgastroentertitis N. Engl. J. Med., 325:152-164, 1991]. Therefore, worldhealth organization (WHO) considered more efficient suppression andprevention for the infection by the rotavirus the first research subject[see reference: Glass, R. I et al., (1994) Rotavirus vaccines: successby reassortment Science 265-1389-1391].

[0003] The rotavirus is usually globular shape and is named after theouter and inner shells or double-shelled capsid structure of the same.The outer capsid is about 70 nm, and inner capsid is about 55 nm indiameter, respectively. The double-shelled capsid of the rotavirussurrounds the core including the inner protein shell and genome. Thegenome of the rotavirus consists of double stranded RNA segmentsencoding at least 11 rotavirus proteins. The inner capsid includes VP6and VP2 proteins. VP4 and VP7 lie in outer side of the double-shelledcapsid and constitute the outer capsid. Depending upon the antigenicityof VP6 which is a group-specific antigen, the rotavirus is divided intoseven groups, A to G. VP2 protein is related to the synthesis of RNA.Group A rotavirus is further divided into the G-type (glycoprotein type)on the basis of the glycoprotein VP7, and P-type (protease-cleavedprotein) on the basis of the VP4 which are associated with an importantimmunogenicity of the virus by forming a neutralizing antibody [seereferences: Estes M. K., et al., (1987) Synthesis and immunogenicity ofthe rotavirus major capsid antigen using a baculovirus expressionsystem, J. Virol. 61:1488-1494; Estes M. K. & Cohen J., (1989) Rotavirusgene structure and function. Microbiol. Rev. 53:410-419; Desselberger U& McCrae M. A., (1994) The rotavirus genome. Curr. Microbiol. Immuno.185:31-66].

[0004] The methods to treat the acute diarrhea disease caused by therotavirus were restricted to administer non-specific physical strengthsupplements such as water and electrolyte. Thus, the vaccine as aneffective therapeutic agent has been required for completely preventingthe human from the all serum-type of human rotavirus. As an attempt, alive vaccine of attenuated human rotavirus, animal rotavirus such ascow, or reassortants comprised of RNA segments derived from differentserotypes human and animal rotavirus have been used. As a result of suchstudy, Wyeth Laboratories, a manufacturing company of vaccines, reportedfirst rotavirus vaccine, RotaShield (trademark). The vaccine wasproduced by reassorting rotavirus of Rhesus monkey and human rotavirusof 3 serum types and was firstly approved by Food and DrugAdministration (FDA) in the world in 1998. However, the FDA's approvalto the vaccine was tentatively canceled in 1999, because of the sideeffects such as intussusception [MMWR Morb. Mortal Wkly. Rep., (1999)5;48(43):1007].

[0005] Apart from the above, a study that the part of rotavirus isproduced on a large scale through genetic recombinant technology and isused as a subunit vaccine is proceeding. In this point, many researchershave studied the expression of the rotavirus capsid proteins in E. coliexpression system, baculrovirus expression system or mammalianexpression system [Smith R. E. et al., (1989) Cloning and expression ofthe major inner capsid protein of SA-11 simian rotavirus in E. coli.Gene 79:239-248; Tosser G. et al., (1992) Expression of the major capsidprotein VP6 of group C rotavirus and synthesis of chimericsingle-shelled particles by using recombinant baculoviruses. J. Virol.66:5825-5831; Ito H. et al., (1997) Expression of the major inner capsidprotein, VP6, of avian rotavirus in mammalian cells. Vet. Microbiol.49:257-265].

[0006] However, it is difficult to produce the rotavirus on a largescale by cell culture, because the rotavirus is infected through themucus cell. That is, because it is difficult to produce the virusprotein particles similar to natural virus in prokaryotic expressionsystems such as E. coli expression system, the particle does not elicitantigenecity. In view of contamination hazard during culture process,purification problem, high cost and low yield, the mammalian expressionsystem is not satisfactory.

[0007] Also, most of disease caused by rotavirus is commonly occurred indeveloping countries which are deficient in sanitary facilities andvaccine supplement. Even if technical success of the method forproducing the virus structural proteins in other cells and the vaccinecomposition comprising the proteins is possible, long time and manystudies are required for commercializing the method in view of highproduction cost and sale price. On the other hand, if the rotavirusstructural protein can be produced by using plant, especially edibleplant and can be used for producing the vaccine, it is very economicalin view of low production cost, no purification process, and efficiencyin transport and storage.

[0008] Therefore, the present inventors found that rotavirus structuralproteins capable of inducing the mucus and systemic immune response canbe produced in the plant cell which is transformed with plant expressionvector including rotavirus capsid gene, and then the plant cell iscultured under the suitable condition. Also, the vaccine compositioncomprising the rotavirus structural proteins can be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIGS. 1a-1 d are cleavage maps showing the structure of binaryvector used for transforming a plant cell, pILTAB357-VP6, pILTAB357-VP2,pILTAB357-VP4 and pILTAB357-VP7,

[0010] CsVMV promoter: Cassava vein mosaic virus promoter

[0011] NOS promoter: nopalin synthase promoter

[0012] NOS 3′: nopalin synthase transcription terminator region

[0013] NPT II: neomycin phosphotransferase II gene

[0014]FIGS. 2a-2 c are the result of western blotting of normal andtransformed tomato cell. The arrows show recombinant rotavirus proteins,VP2, VP4 and VP7 respectively.

[0015] M: marker of molecular weight,

[0016] 1: intracellular fraction of the normal cell,

[0017] 2: medium fraction of the normal cell,

[0018] 3: intracellular fraction of the transformed tomato cell,

[0019] 4: medium fraction of the transformed tomato cell,

[0020] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the examples.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides the structural proteins of therecombinant human rotavirus obtained by culturing transformed plantcells, and the vaccine composition comprising such proteins as aneffective component.

[0022] The method for producing antigenic recombinant human rotavirusstructural protein in plant cell comprising the steps of:

[0023] 1) preparing the recombinant expression plasmid by inserting cDNAfragment encoding human rotavirus structural protein into the expressionplasmid for transforming plant, which comprises the promoter regulatingplant-specific expression and selection marker;

[0024] 2) introducing the recombinant expression plasmid of step 1 intoplant cell;

[0025] 3) inducing formation of callus from the plant cell which isintroduced with the recombinant expression plasmid of step 2;

[0026] 4) regenerating the callus of step 3 to the whole plants; and

[0027] 5) recovering the recombinant human rotavirus structural proteinin the plants of step 4.

[0028] The wide range of sources (e.g., human rotavirus and animalrotavirus such as cow) are available for the gene encoding rotavirusstructural protein of step 1, as long as the gene encodes the rotaviruscapsid protein. And, it is preferable to use the gene encoding innercapsid protein VP6, the gene encoding inner capsid protein VP2, the geneencoding outer capsid protein VP4, and the gene encoding outer capsidprotein VP7. The rotavirus gene of the interest can be obtained byamplifying the gene from the feces of the subject hatingrotavirus-related disease with the known polymerase chain reaction(PCR). In a desired embodiment of the present invention, VP6 genefragment can be available by excising the gene from the plasmid pGEM-VP6(KCTC 8984P). In another embodiment of the invention, cDNA fragmentencoding VP2, VP4, and VP7 serotype G1, VP7 serotype G2, VP7 serotype G3and VP7 serotype G4 can be used by isolating and amplifying the cDNAfragment from the faces of the subject having rotavirus-related disease.

[0029] Also, recombinant plasmid expressing the cDNA fragment encodingthe human rotavirus structural protein of the interest of step 1 can beconstructed from the known plant expression vector as a basic vector.The binary vector, cointegration vector, or a general vector which isdesigned not to include T-DNA region but to be capable of beingexpressed in plant can be also available.

[0030] In the present invention, the examples of the desired binaryvector include final binary vectors, for example, pILTAB357-VP6,pILTAB357-VP2, pILTAB357-VP4, pILTAB357-VP7, which are prepared byinserting the cDNA fragment encoding each human rotavirus structuralproteins, VP6, VP2, VP4 or VP7 into binary vectors comprising leftborder of T-DNA relating lo infection of a foreign gene and right borderof T-DNA for transforming a plant cell, cassava vein mosaic viruspromoter between the left border and the right border, nopalin synthasepromoter, transcription termination region of nopalin synthase, andselection marker for selecting transformants.

[0031] As the promoter regulating the plant-specific expression, all theknown expression vectors maximally expressing the recombinant proteinsin plant cell can be used, and the promoter includes ubiquitin promoter,actin promoter, PG promoter or endosperm-specific promoter other thanthe cassava vein mosaic virus promoter.

[0032] In the transformation of the plant cell in step 2, when thebinary vector or the cointegration vector is used, the plant celltransformation method mediated by agrobacterium will be employed. Insuch case, Agrobacterium tumefaciens or Agrobacterium rhizogenes can beused.

[0033] In the desired embodiment of the present invention, the finalbinary vector can be introduced into Agrobacterium tumefaciens via theknown triparental mating. The tirparental mating is a method ofintroducing the plasmid having the gene of interest to the agrobacteriumby coculturing E. coli including helper plasmid for conjugation. E. coliincluding the recombinant plasmid having the gene of interest andAgrobacterium for transforming the plant cell all together. Morespecifically, the parent E. coli used for triparent mating can be E.coli having the pILTAB357-VP6, and tomato seedling cotyledon can betransformed with the agrobacterium transformed by the tirparentalmating.

[0034] In case of transforming the monocotyledon plant with the vectorwhich does not include T-DNA region, polyethylene glycol-mediateduptake, electroporation, or microparticle bombardment can be used andtransformation using single DNA and simultaneous transformation usingmultiple DNA also can be used.

[0035] The transformed cell can be redifferentiated by using thestandard technologies known to those skilled in the art. The plant cellwhich may be transformed by the above method includes dicotyledon plantsuch as lettuce, Chinese cabbage, radish, potato, and tomato, etc. andmonocotyledon plant such as rice, barley, and banana, etc. Especially,in case of producing the human rotavirus structural protein and theparticle similar to the same by transforming edible plant, uptake of thetransformed plant itself can induce directly the immune response, so itcan be effectively used as a edible vaccine.

[0036] The selection marker to select the transformed plant cell isusually antibiotic resistant gene, but is not limited to the same. Forexamples, herbicide resistant gene, metabolism-related gene, luminousgene, green fluorescence protein (GFP), β-glucuronidase (GUS) gene,β-galactosidase (GAL) gene can be used as selection marker.Specifically, neomycin phosphotransferase II (NPTII) gene, hygromycinphosphotransferase gene, phosphynotricin acetyltransferase gene, ordihydrofolate reductase gene can be used.

[0037] Meanwhile, the transformed plant cell of the present inventioncan be cultured by employing microgravity culture in High AspectRotating-Wall Vessel as well as the suspension culture. When the HighAspect Rotating-Wall Vessel is used, the cell growth rate is very slowcompared to the general suspension culture method, because the lag phaseis longer in order to fit the circumstance of the mimic microgravity.However, it is found that rotavirus protein yield per cell weight in therotary reactor is similar to that in the suspension culture.

[0038] Also, the present invention provides rotavirus-like particlewhich can be made by folding the human rotavirus structural proteins ofthe invention, VP2, VP6, VP4 and VP7. Herein, ‘rotavirus-like particle’is intended to mean the antigenic particle obtained from an assembly ofat least a capsid protein, or self-assembly of the same.

[0039] Furthermore, the present invention provides a vaccine compositionuseful for treatment and prevention from rotavirus-related diseasecomprising the human rotavirus structural protein of the presentinvention as an effective component. More specifically, the vaccinecomposition comprises at least one selected form the group consisting ofVP4, G1 serotype VP7, G2 serotype VP7, G3 serotype VP7, and G4 serotypeVP7, or preferably comprises VP2 and VP6.

[0040] To prepare for the vaccine composition, the human rotavirusstructural proteins produced by the transformed plant cell can beisolated and purified from the cell culture by using the knownpurification method, but in case of transforming by using edible plantcell, it is more preferable to prepare the rotavirus structural proteinsusing plant cell itself, the part of redifferentiated transformed plantor the extract of plant without purification process in view of theproduction cost and process management.

[0041] The vaccine useful for treatment and prevention forrotavirus-related disease can be administered orally or parenterally,and can be used in the usual form of drug.

[0042] Namely, the human rotavirus vaccine composition can be preparedby containing optionally a pharmaceutically acceptable diluent orexcipient such as filler, extender, binding agent, wetting agent,integrant, and surfactant. The solid preparation for oral administrationincludes tablet, pill, powder, grannule, capsule, and can be prepared bymixing at least one of the excipient such as starch, calcium carbonate,sucrose, lactose, and gelatin with rotavirus vaccine composition. Also,lubricant such as magnesium stearate, talc as well as simple excipientscan be used. The liquid preparation for oral administration can containsuspension, liquid drug, emulsion, and syrup, and include many excipient(e.g., wetting agent, sweetening agent, fragrant, preserving agent)other than commonly used simple diluents such as water, liquid andparaffin. The preparation for parenteral administration containssterilized solution, non-aqueous solvent, suspension solvent, isemulsifier, freeze-drying agent or suppository. The non-aqueous solventor suspension solvent can include propylene glycol, polyethylene glycol,vegetable oil such as olive oil, injectable ester such as ethyloleate.The basic material of suppository includes witepsol, macrogol, tween 61,cacao paper, laurin paper, glycerogelatin.

[0043] However, because most of rotavirus vaccine are administered toinfant, especially 2 to 24 month-old infant, it is preferable to beadministered as soon as possible before firstly exposed to therotavirus, and the dosage form of the vaccine is more preferably oralpreparation in liquid, injectable preparation, or suppository in orderto easily administer to the infant.

[0044] As described below, the present invention will be morespecifically illustrated by examples of the expression system that cDNAencoding the recombinant human rotavirus structural protein is expressedin the transformed tomato cell in culture systems, and the expressionsystem that the High Aspect Rotating-Wall Vessel designed by NASA isemployed for optimally culturing the recombinant human rotavirusstructural proteins. However, modification or change of the method canbe used, and thus the method does not limited to the following definiteworking examples.

[0045] Preparation of Vector Including cDNA Fragment Encoding RotavirusStructural Protein

[0046] The gene cloning technology used herein can be easily performedby employing the method known to those skilled in the art. Such methodsare well described in the following references such as Sambrook et al.,1989, Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory, N.Y.; and Ausubel et al., 1989, Current Protocols inMolecular Biology, Greene Publishing Associate and Wiley Interscience,N.Y.

[0047] A kind of binary vector used for transforming dicotyledon plant,especially tomato, is very various, and most of binary vectors can beavailable in international depository authority, for example, Center forthe Application of Molecular Biology to International Agriculture(CAMBIA, GPO Box 3200, Canberra ACT2601, Australia). Basic binary vectorwhich is variously modified to include selection marker, promoter,transcription terminator region in left border and right border regionof transferred gene is used.

[0048] In the examples of the present invention, pGEM-VP6 (KCTC 0944BP)including cDNA encoding human rotavirus VP6 protein was used in order toconstitute the binary vector. Also, the plasmids including cDNA fragmentencoding human rotavirus VP2 of SEQ ID NO: 1, cDNA fragment encodinghuman rotavirus VP4 of SEQ ID NO: 2, cDNA fragment encoding humanrotavirus VP6 of SEQ ID NO: 3, and cDNA fragment encoding humanrotavirus VP7 of SEQ ID NO: 4 to SEQ ID NO: 7 having different serotype,respectively, were used. However, the vector suitable for the presentinvention is not limited to the above plasmids, and all range of thevectors which efficiently express the genes can be used. In the examplesof the present invention, the plasmid transforming plant cell wasconstructed by inserting cDNA fragment to the cloning site of knownplasmid, pILTAB357 (Scripps Research Institute, U.S.A.; Verdaguer et al,Isolation and expression in transgenic tobacco and nice plants, of thecassava vein mosaic virus promoter, Plant Molecular Biology 31:1129-1139, 1996), using CsVMV promoter in T-DNA region of pILTAB357 andinserting neomycin phosphotransferase II (NPTII) gene as a selectionmarker (see FIGS. 1a-1 d).

[0049] Transformation Procedure

[0050] Transformation can be performed according to the method known tothose skilled in the art which the present invention pertains to. Theplant can be transformed via agrobacterium-mediated transformation whichis illustrated in Paszkowsky et al., EMBO J 3:2717-2722 (1984). Forexample, agrobacterium-mediated transformation for the tomato isdescribed in An et al., EMBO J 4-227-288 (1985). The transformation ofmonocotyledon plant can be achieved by directly implanting the gene intoplasma via PEG or electroporation, or by introducing the gene into thecallus tissue via particle bombardment. The transformed cell can beredifferentiated to the whole plant via the standard method known tothose skilled in the art. The host cell for transformation can beusually Agrobacterium tumefaciens, and Agrobacterium tumefaciens strainLBA4404 which is well-known in the art was used in the desiredembodiment of the present invention.

[0051] The binary vector of the, present invention was introduced toAgrobacterium tumefaciens via triparental mating. In such case, forexample, E. coli MM294 including pILTAB357-VP6 which has the insertedcDNA fragment encoding human rotavirus VP6 protein was used as parentstrain. Then, cotyledon excised from tomato (Lycopersicon esclentumMill) seedling was transformed with recombinant Agrobacteriumtumefaciens LBA 4404.

[0052] Transformant Culture Procedure

[0053] To culture the transformed tomato cells, suspension culture andHigh Aspect Rotating-Wall Vessel were used. To do this, sterilizedtomato (Lycopersicon esclentum Mill) seed was inoculated on MS(Murashige and Skoog) medium, and was germinated, and then co-culturedwith suspension culture solution containing Agrobacterium tumefaciensLBA 4404 including recombinant expression plasmid, and culturedsuccessively. Also, to determine the culture system that can optimallyproduce recombinant rotavirus structural proteins, VP6, VP2, VP4 andVP7, the transformed tomato cell was cultured in a microgravity modelsystem with High Aspect Rotating-Wall vessel designed by NASA.

[0054] The Expression of the Recombinant Rotavirus Structural Proteins

[0055] The transformed tomato cell was established from the selectedkanamycin-resistant callus, and was maintained in liquid medium added bykanamycin 200 mg/L for 3 months. The degree of gene expression wasanalyzed using the transformed tomato cell on two weeks after cultureand normal tomato cell. Recombinant rotavirus structural proteins, VP6,VP2, VP4 and VP7 could be detected in the transformed tomato cell bywestern blotting.

[0056] For example, recombinant rotavirus protein VP6 is largely inintracellular fraction, and has molecular weight of about 44 kDa. VP2has molecular weight of 94 kDa, and VP4 has a molecular weight of 87 kDaand VP7 has a molecular weight of 34 kDa. Such measured molecular weightof the proteins is almost consistent with those derived from thenucleotide sequencing. Recombinant rotavirus proteins, VP6, VP2, VP4 andVP7 were not detected in the non-transformed cell or fraction. The factmeans that VP6, VP2, VP4 and VP7 were expressed from pILTAB357-VP6,pILTAB357-VP2, pILTAB357-VP4 and pILTAB357-VP7 transformed in the tomatocell.

[0057] The invention will be further described in the followingexamples, which are not intended to limit the scope of the invention.

EXAMPLE 1

[0058] Construction of Binary Vector for Transformation

[0059] The fragment produced by digesting pGEM-VP6 (KCTC 0944BP)including gene encoding human rotavirus VP6 protein with restrictionenzyme EcoRI was inserted into the EcoRI restriction site of plasmidpILTAB357 (Scripps Research Institute, U.S.A.) to prepare recombinantplasmid pILTAB357-VP6 of 13.94 kb. FIG. 1a shows cleavage map of thepILTAB357-VP6. In the pILTAB357-VP6, the CsVMV promoter inserted betweenthe left border and the right border of binary vector for transformingplant cell, pILTAB357 was used as a promoter for regulating thetranscription of VP6 gene [Verdaguer B., et al., (1996) Isolation andexpression in transgenic tobacco and rice plants, of the cassava veinmosaic virus (CsVMV) promoter. Plant Mol. Biol. 31:1129-1139], andneomycin phosphotransferase II (NPTII) was inserted as a marker forselecting the transformant in the growth medium containing kanamycin(FIG. 1a). By using restriction map and DNA sequencing, the location ofVP6 gene in recombinant plasmid, pILTAB357-VP6, and whether readingframe of the VP6 was correctly inserted were identified. The preparedbinary vector was introduced to Agrobacterium tumefaciens LBA 4404 (Cat.No. 18313-015, GibcoBRL, U.S.A.) via triparental mating [Ainsworth C. etal., (1996) Techniques in Plant Molecular Biology, Wye, UK: WyeCollege]. In the triparental mating, E. coli MM294 (KCCM 70079)including pILTAB357-VP6 was used as parent strain. Cotyledon cut fromtomato (Lycopersicon esclentum Mill) was transformed with therecombinant Agrobacterium tumefaciens LBA 4404 according toagrobacterium-mediated leaf disc transformation [Horsch R. B. et al.,Science 227: 1229-1231, 1985].

EXAMPLE 2

[0060] Transformation Procedure and Establishment of Suspension Culturefor the Transformed Cell

[0061] Tomato (Lycopersicon esculentum Mill) seed was immersed in 70%ethanol solution for 1 minute, sterilized with 1.2% sodium hypochloride(NaOCl) solution for 30 minutes, and then, was washed with sterilizeddistilled water many times. The sterilized seed was inoculated on MSmedium [sucrose 30 g/l, 2,4-D (2,4-dichlorophenoxyacetic acid) 2 mg/l,kinetin 0.2 mg/l, timentin 150 mg/l, pH 5.7] containing agar 8 g/L. Theseed applied on Magenta box was germinated in culture room in lightcycle of 12 hours at 27° C. After about two weeks, germinated sterilizedyoung seedling was used for transformation experiment. Cotyledon excisedfrom the seedling was immersed in suspension culture of recombinantagrobacterium tumefaciens LBA 4404 including pILTAB357-VP6 for 3minutes, and then extra microorganism was removed with filter paper.Then, explants were cultured with agrobacterim tumefaciens LBA 4404 onsolid MS medium containing agar 8 g for 3 days. The resultant explantswere transferred onto MS medium containing kanamycin B 200 mg/L andtimentin 150 mg/l. The explants were incubated in culture room at 27° C.in light cycle of 12 hours for 4 weeks in order to induce callus. Theinduced callus was cut and then was maintained and amplified by passageculture at 4 weeks interval. The callus resistant to kanamycin wasselected and transferred onto the liquid medium containing kanamycin 200mg/L but not agar, and then, was cultured in suspension.

[0062] The transformed plant cell was cultured in suspension mediumcontaining kanamycin B 200 mg/L in rotary shaking flask at 27° C., 100rpm, and was maintained and amplified by passage culture at 3 weeksinterval. The callus of normal tomato cell was induced as controlaccording to all the process described above, except the process oftransformation of agrobacterium, and cultured in suspension under thesame conditions.

EXAMPLE 3

[0063] Suspension Culture and Gene Expression Anaylsis

[0064] 1) Suspension Culture of the Transformed Plant Cell and Isolationof VP6

[0065] The transformed tomato cell was cultured at 27° C., 100 rpm inshaking flask containing MS liquid media 50 mg/L including kanamycin 200mg/L. During the culture time, the cultured cell was collected andanalyzed for the cell growth and expression of the recombinant proteinat a regular interval.

[0066] The culture was centrifugated at 3,000 rpm for 5 minutes toseparate the cell, and supernatant was used for analyzing extracellularrecombinant protein. Cell fraction was freezed with liquid nitrogen, andthen was grinded with mortar and pestle with adding protein extractionsolution [Tris-HCl (pH 7.5) 50 mM, ethylenediaminetetraacetic acid(EDTA) 2 mM, ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA)0.5 mM, phenylmethylsulfonyl fluoride (PMSF) 1 mM, 1% Triton X-100].Then the resultant was sonicated in the centrifugation tube at 4° C. for5 minutes 3 times. The cell extract was centrifuged at 14,000 rpm at 4°C., for 15 minutes to remove cell debris, and supernatant was used foranalyzing the intracellular recombinant protein. Unless especiallydefined, the mixture of intracellular fraction and extracellularfraction was used for analysis of protein production in total.

[0067] 2) Gene Expression Analysis

[0068] The production amount of the recombinant rotavirus VP6 proteinwas measured by western blotting by using fusion VP6 protein of 44-5 kDaexpressed in E. coli as control. The fusion VP6 protein has a molecularweight of 44.5 kDa. The fusion VP6 protein was expressed in E. coliusing pET-15b (Novagen, U.S.A.) designed to express VP6 protein in E.coli and used by purifying with His-Bind kit (Novagen, U.S.A.).Polyhistidine tag sequence linked to cleavage site of thrombin is fusedin N-terminal of fusion VP6 protein.

[0069] According to Laemmli's method, SDS-PAGE electrophoresis wasperformed on the protein sample [Laemmli U.K. (1970) Cleavage ofstructural proteins during the assembly of the head of bacteriophage T4.Nature 227:680-685]. After electrophoresis, the resultant proteins weretransferred onto nitrocellulose membrane and then, were combined withanti-rotravirus polyclonal antibody (Chung-Ang University, Seoul, Korea)obtained from Guinea pig, and were detected by rabbit anti-Guinea pigimmunoglobulin G (Kirkegaard & Perry Laboratories, U.S.A.). Afterwashing nitrocellulose membrane with buffer solution, the resultant wascolored with BM purple AP substrate solution (Boehringer Mannheim,U.S.A.) and quenched by adding distilled water.

[0070] Recombinant rotavirus protein VP6 was largely in intracellularfraction of the transformed plant and had molecular weight of about 44.5kDa, which is consistent with that of VP6 expressed in E. coli. Thisshows that the transformed plant cell of the present invention expressedsuccessfully rotavirus protein VP6.

[0071] The production amount of recombinant rotavirus proteins VP6 was0.33 mg/L on 18 day after culture.

EXAMPLE 4

[0072] Microgravity Culture

[0073] To test microgravity, culture solution of the transformed tomatocell wasa filled up in High Aspect Rotating-Wall vessel (HARV,Synthecon, Houston, Tex.) with the capacity of 10 ml to producerecombinant rotavirus VP6 and cultured for 18 days. The productionamount of recombinant rotavirus VP6 in HARV was 0.13-0.15 mg/L.

EXAMPLE 5

[0074] Except that recombinant plasmid, pILTAB357-VP2 of 15.0 kbprepared by using pGEM-VP2 (KCTC 0947BP) including cDNA fragment of SEQID NO: 1 encoding human rotavirus protein VP2, was used, instead ofpGEM-VP6 (KCTC 0944BP) including cDNA fragment encoding human rotavirusprotein VP6, cotyledon excised from tomato seedling was transformed withAgrobacterium tumefaciens to which introduced binary vectorpILTAB357-VP2 according to the same method of Examples 1-3. Then,transformed tomato cell was cultured in suspension. The recombinantrotavirus protein VP2 was isolated, and was analyzed with westernblotting in order to identify that the transformed cell expressesrotavirus protein VP2 successfully (FIG. 2a).

[0075] pGEM-VP2 including cDNA fragment encoding VP2 derived from humanrotavirus was deposited on Feb. 8, 2001 to Korea Collection for TypeCulture (KCTC) under Budapest treaty on the international recognition ofthe deposit of microorganism, and was acceded KCTC 0947BP as a depositnumber. The cleavage map of recombinant plasmid, pILTAB357-VP2 was shownin FIG. 1b.

EXAMPLE 6

[0076] Except that recombinant plasmid, pILTAB357-VP4 of 14.8 kbprepared by using pGEM-VP4 (KCTC 0945BP) including cDNA fragment of SEQID NO: 2 encoding human rotavirus protein VP4, was used, instead ofpGEM-VP6 (KCTC 0944BP) including cDNA fragment encoding human rotavirusprotein VP6, cotyledon excised from tomato seedling was transformed withAgrobacterium tumefaciens to which introduced binary vectorpILTAB357-VP4 according to the same method of Examples 1-3. Then,transformed tomato cell was cultured in suspension. The recombinantrotavirus protein VP4 was isolated, and was analyzed with westernblotting in order to identify that the transformed cell expressesrotavirus protein VP4 successfully (FIG. 2b).

[0077] pGEM-VP4 including cDNA fragment encoding VP4 derived from humanrotavirus was deposited on Feb. 8, 2001 to Korea Collection for TypeCulture (KCTC) under Budapest treaty on the international recognition ofthe deposit of microorganism, and was acceded KCTC 0945BP as a depositnumber. The cleavage map of recombinant plasmid, pILTAB357-VP4 was shownin FIG. 1c.

EXAMPLE 7

[0078] Except that recombinant plasmid, pILTAB357-VP7 of 13.7 kbprepared by using pGEM-VP7 (KCTC 0946BP) including cDNA fragment of SEQID NO: 4 encoding human rotavirus protein VP7 serotype G1, was used,instead of pGEM-VP6 (KCTC 0944BP) including cDNA fragment encoding humanrotavirus protein VP6, cotyledon excised from tomato seedling wastransformed with Agrobacterium tumefaciens to which introduced binaryvector pILTAB357-VP7 according to the same method of Examples 1-3. Then,transformed tomato cell was cultured in suspension. The recombinantrotavirus protein VP7 was isolated, and was analyzed with westernblotting in order to identify that the transformed cell expressesrotavirus protein VP7 serotype G1 successfully (FIG. 2c).

[0079] pGEM-VP7 including cDNA fragment encoding VP7 having serotype G1derived from human rotavirus was deposited on Feb. 8, 2001 to KoreaCollection for Type Culture (KCTC) under Budapest treaty on theinternational recognition of the deposit of microorganism, and wasacceded KCTC 0946BP as a deposit number. The cleavage map of recombinantplasmid, pILTAB357-VP7 was shown in FIG. 1d.

EXAMPLE 8

[0080] The pCR2.1-VP7 (KCTC 0948BP) including cDNA fragment of SEQ IDNO: 5 encoding human rotavirus protein VP7 serotype G2 was used, insteadof pGEM-VP7(KCTC 0946BP) including cDNA fragment encoding humanrotavirus protein VP7 serotype G1 as described in Example 7. It wasconfirmed that the transformed cell expressed rotavirus protein VP7serotype G2 successfully by working according to the same method asexample 7.

[0081] The pCR2.1-VP7 including cDNA fragment encoding VP7 serotype G2derived from human rotavirus was deposited on Feb. 8, 2001 to KoreaCollection for Type Culture (KCTC) under Budapest treaty on theinternational recognition of the deposit of microorganism, and wasacceded KCTC 0948BP as a deposit Number.

EXAMPLE 9

[0082] The pCR2.1-VP7 (KCTC 0949BP) including cDNA fragment of SEQ IDNO: 6 encoding human rotavirus protein VP7 serotype G3 was used, insteadof pGEM-VP7 (KCTC 0946BP) including cDNA fragment encoding humanrotavirus protein VP7 having G1 serotype as described in Example 7. Itwas confirmed that the transformed cell expressed rotavirus protein VP7serotype G3 successfully by working according to the same method asexample 7.

[0083] The pCR2.1-VP7 including cDNA fragment encoding VP7 serotype G3derived from human rotavirus was deposited on Feb. 8, 2001 to KoreaCollection for Type Culture (KCTC) under Budapest treaty on theinternational recognition of the deposit of microorganism, and wasacceded KCTC 0949BP as a deposit Number.

EXAMPLE 10

[0084] The pCR2.1-VP7 (KCTC 0950BP) including cDNA fragment of SEQ IDNO: 7 encoding human rotavirus protein VP7 serotype G4 was used, insteadof pGEM-VP7 (KCTC 0946BP) including cDNA fragment encoding humanrotavirus protein VP7 having G1 serotype as described in Example 7. Itwas confirmed that the transformed cell expressed rotavirus protein VP7serotype G4 successfully by working according to the same method asexample 7.

[0085] The pCR2.1-VP7 including cDNA fragment encoding VP7 serotype G4derived from human rotavirus was deposited on Feb. 8, 2001 to KoreaCollection for Type Culture (KCTC) under Budapest treaty on theinternational recognition of the deposit of microorganism, and wasacceded KCTC 0950BP as a deposit Number.

[0086] Industrial Applicability

[0087] According to the genetic recombination technology of the presentinvention, the method for producing rotavirus structural proteins inplant cell on a large scale and vaccine composition comprising theproteins as an effective component has advantages in low production costand no need to purification. Also, when the protein is produced fromedible plant, it is possible to use the protein as edible vaccine ororal vaccine by preparing vaccine comprising the isolated virusstructural proteins and pharmaceutically acceptable additive, or bytaking the plant itself directly. In particular, when it is important toinduce mucus immune response such as rotavirus, the oral vaccinecontributes desired mucous and systemic immune response. Thus, theprotein can be used for vaccine composition useful for treatment andprevention from rotavirus-related disease. In addition, the method ofthe present invention is easy and efficient in aspect of transport andstorages compared to other culture system.

1 7 1 2729 DNA Human rotavirus 1 tcctattaaa ggttcaatgg cgtacaggaagcgtggagct aaacgtgaag acttactaca 60 acaaaatgaa cgtctgcaag aaaaagaaattgaaaataat accgacgtaa ccatggaaaa 120 tagaaataaa aataaaaata acaatagaaagcagcaatta tctgacaaag tgttatcacg 180 aaaagaggaa ataataactg atgtgcaagatgacattaaa atagctgatg aggtcaaaaa 240 atcatcaaaa gaagagtcga aacagttacttgaaatatta aaaacgaaag aagatcatca 300 gaaagaagta cagtacgaaa ttttacaaaaaacaatacca acttttgaac caaaagaatc 360 aattctgaaa aaattagaag atataagaccagaacaagct aagaagcaaa tgaaattgtt 420 tagaatattt gaaccaagac aattaccaatctatcgagca aacggtgaga aagaattaag 480 gaatagatgg tactggaaat taaaaaaggatacactacca gacggagatt atgacgtacg 540 agaatatttt ttaaatttat atgatcaaatactgatagaa atgccagatt atttattgtt 600 aaaagatatg gctgtagaaa ataaaaactccagggatgct ggtaaggttg tagattctga 660 gactgcaagt atttgtgatg ctatatttcaagatgaggag acagagggag ttattagaag 720 atttatagca gatatgagac aacaagttcaggctgataga aatattgtca attatccatc 780 aattttacat ccaattgatt atgcatttaatgaatatttt ctaaaccatc aattagttga 840 accattgaat aacgaaatta tttttaattatataccagaa agaataagga atgatgtcaa 900 ttatattttg aatatggata tgaatttaccatcaacagca agatatatta gaccaaatct 960 attgcaagat agactgaatt tacatgataattttgagtca ttatgggaca caataactac 1020 atcaaattac atattggcaa gatcagtcgtgcctgatttg aaggaaaaag aattagtttc 1080 aactgaagct caaatacaga aaatgtctcaagatttacag cttgaagctt taacaataca 1140 atctgaaacg cagtttctag ccggtataaattcacaagca gcgaatgatt gttctaaaac 1200 attgatagca gctatgttaa gccaacgtacaatgtcatta gattttgtaa ctacgaatta 1260 tatgtcactt atatctggca tgtggctattgactgttata ccaaatgaca tgtttcttcg 1320 cgagtcgcta gtcgcatgcg aactggctataataaatact atagtttacc cagcatttgg 1380 aatgcaaaga atgcattata gaaatggcgatccccagact ccatttcaaa tagcagaaca 1440 acaaatacaa aatttccaag tagctaattggttacatttt attaataata atagatttag 1500 acaagtcgtt attgatggag tgttaaatcaaacacttaac gacaacatta ggaatggaca 1560 agttattaat caattaatgg aagcattaatgcaattacct agacaacaat ttccgaccat 1620 gccagttgat tataaaagat caatccaaagaggaatatta ttattgtcta acagattagg 1680 tcagttagtt gatttaacaa gattattatcatataattat gaaactctaa tggcttgtat 1740 aactatgaat atgcagcatg ttcaaactctcactaccgaa aaattacagt taacttctgt 1800 cacatcttta tgtatgttaa ttggaaatactacagtaatt ccaagcccac aaacactatt 1860 tcactattat aacgtaaatg taaattttcattcaaattat aacgaacgaa ttaacgatgc 1920 tgtagctatt attacggctg ctaatagactaaacttatat cagaaaaaaa tgaaatcaat 1980 agttgaggat tttttgaaaa ggttgcaaatttttgatgta ccacgagtac cagacgacca 2040 aatgtacagg ttgagagaca gacttagattattgccagtt gaaagacgaa gacttgatat 2100 atttaactta atattaatga atatggagcagatcgaacga gcttcagata aaattgctca 2160 aggagtaata attgcttaca gagatatgcagctagaaaga gatgagatgt atggatttgt 2220 taacattgct aggaacctcg atggatatcaacaaatcaat ttagaagagt taatgagaac 2280 tggagattat ggtcagatta ctaatatgctactaaacaac cagcctgtag ctttggttgg 2340 agcattgcca tttatgacag attcatcagttatatcgctc attgcaaaat tggatgccac 2400 agtttttgct caaatagtta aacttagaaaagtggacact ttaaaaccaa tattatataa 2460 aataaattcc gattctaatg atttctacttagttgcaaat tatgattgga taccaacttc 2520 aaccacaaaa gtctataagc aagtaccacaaccttttgat ttcaggacgt caatgcatat 2580 gttaacgtct aatttaactt ttaccgtttactctgatttg ctatcttttg tttctgcaga 2640 cacggttgaa cctattaacg caattgcttttgacaatatg cgcattatga acgaactgta 2700 aacgccaacc ccattttgga gatatgccg2729 2 2360 DNA Human rotavirus 2 ggctataaaa tggcttggca catttatagacaacttctca ctaattcata ctcagtagat 60 ttacatgatg aaatagaaca aattgggtcagaaaaaactc aaaacgtaac tgtaaatcca 120 ggtccgttcg ctcaaactag gtacgctccagttaattggg gtcatggaga gataaatgat 180 tcaaccacag tagaaccaat tttagacggtccttatcagc ctactacatt tacaccacct 240 actgattatt ggatacttat taactcaaatacaagtggag tagtatacga gagtacgaat 300 aatagtgact tttggactgc agtcgtcgctgttgaacctc acgttaatcc agtagataga 360 caatacactg tatttggtga aaataaacaatttaatgtaa gaaatgattc agataagtgg 420 aagtttttag aaatgtttag aagcagtagtcaaaatgaat tttataatag acgtacacta 480 acttctgata ctaaactcgt gggaatattaaagtatggtg gaaggatatg gacgtttcat 540 ggtgaaacac cgagagctac tactgatagttcaaatacta caaatttaaa cgatatatca 600 attataatac attcagagtt ttatattattccaaggtctc aagaatctaa gtgtaatgag 660 tatattaata atggtttacc accaattcaaaatactagaa atgtagtacc attatcatta 720 tcatctagat ccatacagta taagagagctcaagttaatg aagacattac aatttcgaaa 780 acctcattat ggaaagagat gcaatataatagggatatta taattagatt taaatttggt 840 aatagtgtta taaaactggg aggactaggttataaatggt ccgaaatatc atttaaggca 900 gcaaactatc aatataatta tttacgtgatggcgaacaag taactgcaca tactacttgc 960 tcagtaaatg gagtaaataa ttttagctataacgggggat ctctacctac taattttagc 1020 atctcaagat atgaagttat caaagaaaattcttatgtat atgtagatta ttgggatgat 1080 tcaaaagcgt ttagaaatat ggtatatgtcagatcattgg cagctaattt aaactcggtg 1140 aaatgtacag gtggaagtta cgactttagtatacctgtag gtgcatggcc agttatgaat 1200 ggaggcgctg tttcgttaca ttttgctggagttacattat ctacgcaatt cacagatttc 1260 gtatcattga attcattacg atttagatttagtctgacag tggatgaacc atctttttca 1320 atattgagaa cacgtacagt gaatttgtacgggttaccag cagctaatcc aaataatgga 1380 aatgaatact acgaaatatc aggaaggttttcgctcattt ctttagttcc aactaatgat 1440 gattatcaga ctccaattat gaattcagtaacagtaagac aagatttaga acgtcaactt 1500 actgatttac gagaggaatt caattcattatcacaagaaa tagctatgtc acaattaatt 1560 gatttagcat tattaccttt agatatgttttctatgttct caggaatcaa aagtacaatt 1620 gatctgacta aatcgatggc aactagtgtaatgaaaaaat ttagaaaatc aaaattagct 1680 acatcaattt cggaaatgac taattcattatcagacgcag catcatcggc atcaagaagc 1740 gtttccatca gatcgaatat atccacaatttcgaatttga ctaacgtttc aaatgatgta 1800 tcaaatgtga ctaatgcgtt gaatgatatttcaacacaaa catctacaat cagtaagaaa 1860 cttagattaa gagaaatgat tactcaaactgaaggaatga gttttgatga tatttcagcg 1920 gcagtattaa aaacaaaaat agatatgtctactcaaattg gaaagtatac tttacccgac 1980 atagtcacag aggcatctga gaaatttattccaaaacgat catatcgaat attaaaagat 2040 gacgaagtga tggaaattaa tactgaagggaaagtctttg catataaaat agatacactt 2100 aatgaagtac cattcgacgt aaataaatttgctgaccttg taacaaattc tccagttata 2160 tcagcaataa tagattttaa aacattaaaaaacttgaatg ataattatgg aattactcgg 2220 atagaagcac taaatttaat taaatcgaatccaaatgtat tacgtaattt cattaaccaa 2280 aataatccaa ttataaagaa tagaattgaacagctaattc tacaatgtaa gttgtgagaa 2340 tgcttctgga ggatgtgacc 2360 3 1356DNA Human rotavirus 3 ggcttttaaa cgaagtcttc gacatggagg ttctgtactcattgtcaaaa actcttaaag 60 atgccagaga taagattgtt gaaggtacat tatattctaatgttagcgat ctcattcagc 120 aatttaatca aatgatagta accatgaatg gaaatgactttcaaaccgga ggaattggca 180 atttacctgt tagaaactgg acattcgact ttggtctattaggtactaca cttttaaatc 240 ttgatgccaa ctatgttgag actgcaagaa ctacgattgagtattttatt gactttattg 300 ataatgtatg tatggatgaa atggcaagag agtctcaaagaaatggagta gctccacaat 360 ctgaggcact gaggaagcta gccggtatta aatttaaaagaataaatttt aataattcat 420 cagattatat agaaaattgg aatttgcaaa atagaagacaacgtactgga tttgtttttc 480 ataaacctaa tatatttcca tactcagcat catttactttaaatagatct caaccgatgc 540 atgataattt gatgggaacc atgtggctta atgctggatcagaaattcaa gtggctggat 600 ttgactactc gtgtgctcta aatgctccag caaatattcagcagtttgaa catgttgtcc 660 agcttaggcg tgcgctaact acagctacta taactttgttacctgatgca gaaagattta 720 gttttccaag agttattaat tcagcagatg gcgcaactacatggttcttt aatccaatta 780 ttctgagacc aaacaatgta gaggtagaat ttttacttaatggacaaatt attaatacat 840 atcaagctag atttggcact attatcgcaa gaaattttgatacaattcgt ctatcatttc 900 aattaatgcg tccaccaaac atgacaccag ctgtaaatgcattatttccg caagcacaac 960 cttttcaaca tcatgcaaca gttggactta cgttacgtattgattctgca gtttgtgaat 1020 cagtgcttgc ggatgcaaat gaaactttat tggcaaatgttaccgcagta cgccaagagt 1080 atgctatacc agttggccca gtattcccac caggtatgaattggactgag ctgattacta 1140 attattcacc atctagggaa gataatttgc aacgtgtttttacagtagcc tctatcagaa 1200 gcatattgat taagtgagga ccagactaac catctggtatccaatcttag ttagcatgta 1260 gctacttcaa gtcattcaga ctctgcaagt aaggacatgatttcatgttc gctacgtaga 1320 gtaactgcat gaatgatgta gtgagaggat gtgccc 13564 1062 DNA Human rotavirus 4 ggctttaaaa gcgagaattt ccgtctggct aacggttagctccttttgat gtatggtatt 60 gaatatacca caattctaat ctttctgata tcaatcattctacttaacta tatattaaaa 120 tcagtgaccc gaataatgga ctacattata tatagatttttgttaatttc tgtagcatta 180 tttgccttga ctagagctca gaactatgga cttaatataccaataacagg atcaatggac 240 actgtatata tcaactctac tcaagaagga atgtttctaacatccacatt atgtttgtat 300 tatccaactg aagcaagagc tcaaatcagt gatggtgaatggaaagactc attatcacaa 360 atgtttctta caaaaggttg gccaacagga tcagtctattttaaagagta ctcaaatatt 420 gttgattttt ccgttgaccc acaattatat tgtgattataacttagtact aatgaagtat 480 gatcaaaatc ttgaactaga tatgtcagaa ttagccgatttgatattgaa tgaatggtta 540 tgtaatccaa tggatgtaac attatattat tatcaacaatcgggagaatc aaataagtgg 600 atatcgatgg gatcatcatg tactgtaaag gtgtgtccgttgaatacaca aacgttagga 660 ataggttgtc aaacaacgaa tgtagactca tttgaaacagttgctgaaaa tgaaaaattg 720 gctatagtgg atgtcgttga tggaataaat cataaaataaatttgacaac tacgacatgt 780 actattcgaa attgtaagaa gttaggtcca agagagaatgtagctgtcat acaagttggt 840 ggctctaata tattagacat aacagcggat ccaacgactaatccacaaat tgagagaatg 900 atgagagtga attggaaaag atggtggcaa gtattttatactatagtaga ttatattaat 960 cagattgtac aggttatgtc caaaagatca agatcattaaattctgctgc gttttattat 1020 agagtataga tatatcttag attagagttg tatgatgtgacc 1062 5 1062 DNA Human rotavirus 5 ggctttaaaa gcgagaattt ccgtctggctagcggttagc tctttttaat gtatggtatt 60 gaatatacca caattctgac cattttgatatttatcatat tattgaatta tatattaaaa 120 actataacta acacgttgga ctatataatttttaggcttt tactactcat cgctctgatg 180 tcaccatttg tgaggacgca aaattatggtatgtatttac caataacagg atcaccagac 240 gctgtataca caaattcaac tagtggagaatcatttctaa cttcaacgct atgtttatac 300 tatccaacag aagctaaaaa tgagatttcagataatgaat gggaaaatac tctatcgcaa 360 ttatttttaa ctaaaggatg gccgactggatcagtttatt ttaaagacta caatgatatt 420 actacatttt ctatgaatcc acaactatattgtgattata atgtagtatt aatgagatat 480 gataatacat ctgaattaga tgcatcggagttagcagatc ttatattgaa cgaatggctg 540 tgcaatccta tggatatatc actttactattatcaacaaa atagcgaatc aaataaatgg 600 atatcaatgg gaacagactg tacggtaaaagtttgtccac tcaatacaca aactttagaa 660 attgaatgca aaaatacgaa cgtggatacatttgagattg ttgcctcgtc tgaaaaattg 720 gtaattactg atgttgtaaa tggtgttaatcataaaataa atatttcaat aagtacgtgt 780 actatacgta attgtaataa actaggaccacgagaaaatg ttgctataat tcaagttggt 840 ggaccgaacg cactagatat cactgctgatccaacaacag ttccacaggt tcaaagaatc 900 atgcgagtaa attggaaaaa atggtggcaagtgttttata cagtagttga ctatattaac 960 caaattatac aagttatgtc caaacggtcaagatcattag acacagctgc tttttattat 1020 agaatttaga tatagctttg gttagagttgtatgatgtga cc 1062 6 1062 DNA Human rotavirus 6 ggctttaaaa gagagaatttccgtctggct agcggttagc tccttttaat gtatggtatt 60 gaatatacca cagttttaacctttttgata tcagttatat tgttgaatta cgtactcaaa 120 tccttaacta gaataatggactttattatt tacagatttc ttttgattat agttatatta 180 tcaccattcc ttaatgcacaaaattatgga ataaatcttc cgattactgg ctcaatggac 240 acaccatata tgaactcaacacgagaggaa gtattcctaa cttcgacttt atgtttgtat 300 tacccaactg aagcagcaacagaaataaat gataattcat ggaaggatac actttctcag 360 ctatttttaa tcaaaggatggccaacagga tctgtttatt ttaaagatta tactgatatt 420 gcctcgtttt cagttgatccacaactgtac tgtgattata atttggtatt aatgaaatac 480 gacgctacac tgcaactggacatgtccgaa ctagcagatt tgttacttaa tgaatggtta 540 tgtaatccta tggatattactttgtattat tatcaacaaa ctgatgaggc aaataagtgg 600 atttcaatgg gatcatcttgtactataaag gtatgtccac taaatacgca aacattagga 660 attgggtgcc taacaactgatacaaacacg tttgaagaag ttgcaacagc tgaaaaatta 720 gtgattactg acgttgtagatggcgtcaat cataaattga acgtgacgac aaacacttgt 780 acgattagaa attgtaaaaaattaggacca agggaaaacg tagcagttat acaggttggt 840 ggcccagatg tacttgacataacagctgat ccaacgacaa tgccacaaac agaaagaatg 900 atgcgagtga attggaagaaatggtggcaa gtgttttata caatagttga ctacgtgaat 960 caaattgtgc aagcgatgtccaaaagatcg agatcattaa attctgctgc attttactac 1020 agggtatagg tatagcttaggttagaattg tatgatgtga cc 1062 7 1062 DNA Human rotavirus 7 ggctttaaaagcgcgaattt ctgtttggct agcagatagc tccttttaat gtatggtatt 60 gaatataccacagttctatt ttatttgata tcgttcgttc ttgtgagtta tattctgaaa 120 actataataaagataatgga ctatattatt tatagaataa catttgtaat tgtagtatta 180 tcagtattatcgaatgcaca aaattatgga ataaatttgc caattactgg atctatggat 240 acagcatatgctaactcaac acaagacaat aattttttat cttcaacttt atgtctatat 300 tatccatcagaagctccaac tcaaattagt gacactgaat ggaaagatac actatctcag 360 ttgtttttaaccaaaggatg gccgacaggt tcagtttatt ttaatgaata ttcaaacgtt 420 ttagaattttccatcgaacc aaagttatac tgtgattata atgttgtgct gattaagttc 480 gcttctggtgaggaattgga catatctgaa ttagctgatc taatattgaa tgagtggtta 540 tgtaatccaatggatataac attatattat tatcagcaaa ctggagaggc gaataaatgg 600 atatcaatgggatcatcatg taccgttaaa gtgtgtccat taaatactca gacattagga 660 attggatgtcaaacgacaaa tacagctact tttgaaacag ttgctgatag cgaaaaattg 720 gcaataattgatgttgtcga cagtgtaaat cataaattag atgttacatc tactacatgt 780 acaatacggaattgtaataa actaggaccg agagaaaatg tggccataat acaggttggc 840 ggttctaatatattagatat aacagctgat cccacaactt ctccacaaac agaacgaatg 900 atgcgcgtgaattggaaaaa atggtggcaa gtattctata ctgtagttga ttacattaat 960 cagatagtacaagtaatgtc caaaagatcg agatcgttag attcgtcatc tttctattat 1020 agagtgtagatatatcctaa aatagagttg tatgatgtga cc 1062

What is claimed is:
 1. (amended) A method for producing antigenicrecombinant human rotavirus structural protein in plant cell comprisingthe steps of: 1) preparing the recombinant expression plasmid byinserting cDNA fragment encoding human rotavirus structural protein intothe expression plasmid for transforming plant, which comprises thepromoter regulating plant-specific expression and selection marker; 2)introducing the recombinant expression plasmid of step 1 into plantcell; 3) inducing formation of callus from the plant cell which isintroduced with the recombinant expression plasmid of step 2; 4)regenerating the callus of step 3 to the whole plants; and 5) recoveringthe recombinant human rotavirus structural protein in the plants of step4.
 2. (canceled)
 3. (amended) The method according to claim 1, whereinthe human rotavirus structural protein of step 1 is VP2, VP4, VP6 orVP7.
 4. The method according to claim 1, wherein the cDNA fragmentencoding rotavirus structural protein has the sequence represented bySEQ ID NO: 1 encoding VP2 derived from human rotavirus.
 5. The methodaccording to claim 1, wherein the cDNA fragment encoding rotavirusstructural protein has the sequence represented by SEQ ID NO: 2 encodingVP4 derived from human rotavirus.
 6. The method according to claim 1,wherein the cDNA fragment encoding rotavirus structural protein has thesequence represented by SEQ ID NO: 3 encoding VP6 derived from humanrotavirus.
 7. The method according to claim 1, wherein the cDNA fragmentencoding rotavirus structural protein has the sequence represented bySEQ ID NO: 4 encoding VP7 serotype G1 derived from human rotavirus. 8.The method according to claim 1, wherein the cDNA fragment encodingrotavirus structural protein has the sequence represented by SEQ ID NO:5 encoding VP7 serotype G2 derived from human rotavirus.
 9. The methodaccording to claim 1, wherein the cDNA fragment encoding rotavirusstructural protein has the sequence represented by SEQ ID NO: 6 encodingVP7 serotype G3 derived from human rotavirus.
 10. The method accordingto claim 1, wherein the cDNA fragment encoding rotavirus structuralprotein has the sequence represented by SEQ ID NO: 7 encoding VP7serotype G4 derived from human rotavirus.
 11. (amended) The methodaccording to claim 1, wherein the promoter regulating plant-specificexpression of step 1 is cassava vein mosaic virus promoter, ubiquitinpromoter, actin promoter, PG promoter, or endosperm-specific promoter.12. The method according to claim 1, wherein the selection marker ofstep 1 is neomycin phosphotransferase II (NPTII) gene, hygromycinphosphotransferase gene, phosphynotricin acetyltransferase gene, ordihydrofolate reductase gene.
 13. The method according to claim 1,wherein the plasmid of step 1 comprises sequentially the right border ofT-DNA, nopalin synthase promoter, neomycin phosphotransferase II (NPTII) gene, nopalin transcription termination region, CaMV 35S promoter,cDNA fragment encoding rotavirus structural protein, nopalintranscription termination region and the left border of T-DNA. 14.(amended) The method according to claim 13, the recombinant expressionplasmid of step 1 is plasmid pILTAB357-VP6 as shown in the cleavage mapof FIG. 1a, plasmid pILTAB357-VP2 as shown in the cleavage map of FIG.1b, plasmid pILTAB357-VP4 as shown in the cleavage map of FIG. 1c, orplasmid pILTAB357-VP7 as shown in the cleavage map of FIG. 1d. 15.(amended) The method according to claim 1, wherein the introduction ofthe recombinant expression plasmid into plant cell of step 2 isperformed by culturing the plant cell and the agrobacterium transformedwith recombinant binary vector or cointegration vector together.
 16. Themethod according to claim 15, wherein the agrobacterium is agrobacteriumtumefacience or agrobacterium rizogenes.
 17. (amended) The methodaccording to claim 1, wherein the introduction of the recombinantexpression plasmid into plant cell of step 2 is performed byPEG-mediated uptake, microparticle bombardment, or electroporation. 18.The method according to claim 1, wherein the plant cell of step 2 isedible plant cell selected from the group consisting of tomato, lettuce,Chinese cabbage, banana, potato, radish and rice.
 19. (amended) Arecombinant human rotavirus structural protein produced by the methodaccording to any one of claims 1 to
 18. 20. (amended) A rotavirus-likeparticle prepared by assembling after folding recombinant humanrotavirus proteins, VP2, VP6, VP4, and VP7 produced by the methodaccording to any one of claims 1 to
 18. 21. (amended) A rotavirus-likeparticle prepared by assembling after folding recombinant humanrotavirus proteins, VP2, VP4, and VP7 produced by the method accordingto any one of claims 1 to
 18. 22. (amended) A vaccine composition forprevention and treatment of rotavirus-related disease comprising therecombinant human rotavirus structural protein produced by the methodaccording to any one of claims 1 to 18 as an effective component. 23.(amended) The vaccine composition according to claim 22, wherein therecombinant human rotavirus structural protein is any one or moreselected from the group consisting of VP2, VP4, VP6, VP7 serotype G1,VP7 serotype G2, VP7 serotype G3, or VP7 serotype G4.
 24. (amended) Thevaccine composition according to claim 22, wherein the recombinant humanrotavirus structural protein exists in the form of plant cell itself,transgenic plant redifferentiated from plant cell, or the extract ofplant cell.
 25. The vaccine composition according to claim 22, whereinthe vaccine is administered orally or edibly for immunization. 26.(added) A transformed tomato comprising cDNA fragment encoding humanrotavirus structural protein.
 27. (added) The transformed tomatoaccording to claim 26, wherein the cDNA fragment encoding rotavirusstructural protein has the sequence represented by SEQ ID NO: 1 encodingVP2 derived from human rotavirus.
 28. (added) The transformed tomatoaccording to claim 26, wherein the cDNA fragment encoding rotavirusstructural protein has the sequence represented by SEQ ID NO: 2 encodingVP4 derived from human rotavirus.
 29. (added) The transformed tomatoaccording to claim 26, wherein the cDNA fragment encoding rotavirusstructural protein has the sequence represented by SEQ ID NO: 3 encodingVP6 derived from human rotavirus.
 30. (added) The transformed tomatoaccording to claim 26, wherein the cDNA fragment encoding rotavirusstructural protein has the sequence represented by SEQ ID NO: 4 encodingVP7 derived from human rotavirus.
 31. (added) The transformed tomatoaccording to claim 26, wherein the cDNA fragment encoding rotavirusstructural protein has the sequence represented by SEQ ID NO: 5 encodingVP7 derived from human rotavirus.
 32. (added) The transformed tomatoaccording to claim 26, wherein the cDNA fragment encoding rotavirusstructural protein has the sequence represented by SEQ ID NO: 6 encodingVP7 derived from human rotavirus.
 33. (added) The transformed tomatoaccording to claim 26, wherein the cDNA fragment encoding rotavirusstructural protein has the sequence, represented by SEQ ID NO: 7encoding VP7 derived from human rotavirus.